Anandamide (AEA) is a naturally occurring ligand that binds to cannabinoid receptors and is widely distributed in the body, particularly the nervous system. Although AEA was described in 1992, the pathway for its synthesis has not been definitively determined. Three pathways have been published that all start from a single precursor called N-arachidonoyl-phosphatidylethanolamine (NArPE). The first pathway utilizes one enzyme called NAPE-PLD to form AEA. A second pathway has three steps that utilize two enzymes called ??? hydrolase 4 (Abhd4) and a phosphodiesterase called GDE1. The third pathway has two enzymes (phospholipase C and a phosphatase) and has phospho-AEA (pAEA) as an intermediate. In all three pathways intermediates have been identified and the pathways have been shown to be valid when exogenous substrates are added to tissue homogenates or to cells in culture. A conventional mouse knockout (called NAPE-PLD-/-) has been characterized and indicated that the second pathway was dominant in AEA production in brain. However, this conventional NAPE-PLD knockout may introduce compensatory changes in anandamide's biosynthetic pathway(s) during embryogenesis and development and these changes may drive the synthesis of anandamide by one of the parallel pathways. To circumvent the major limitation of a conventional NAPE-PLD-/-, we propose to develop an adult conditional NAPE-PLD knockout by breeding mice in which the exon encoding the enzyme's catalytic residues is floxed. These mice will also be transgenic for a Cre recombinase gene that is under the control of a tetracycline operon that can be turned on in the absence of doxycycline. The tetracycline transactivator will be under the control of a neuron-specific enolase promoter so that -/- NAPE-PLD will be restricted to the nervous system. In this model the ablation of NAPE-PLD will occur during the adult stage in the nervous system, so that compensatory pathways cannot take effect. The brains from the control and experimental animals will be analyzed for AEA and NArPE, their respective congeners as well as pAEA. The NAPE-PLD pathway is the only one that shows calcium dependence, a property one would expect for a postsynaptic enzyme involved in AEA synthesis. The trigger for AEA synthesis would be an increase in postsynaptic calcium resulting from presynaptic neurotransmitter release. Understanding the synthesis of anandamide is essential owing to its effects upon movement, memory, nociception, endocrine regulation, thermoregulation, sensory perception, cognitive functions, mood, and addictive behavior for opiates, alcohol and nicotine. PUBLIC HEALTH RELEVANCE: Maintenance of the levels of anandamide, an endocannabinoid neurotransmitter, is required for normal physiological functioning in humans. The steps by which anandamide is synthesized in the body is the focus of this proposal and may lead to new targets for pharmaceutical drugs. Since anandamide is involved in such diverse functions as reinforcement, mood, memory, appetite, pain and movement, understanding how it is synthesized may lead to treatment for drug addiction, depression, compulsive behaviors, neuropathic pain and movement disorders.